Purpose: It is well established that ionizing radiation of cells produces intracellular H2O2 molecules in cells which in turn induces cell death in the forms of apoptosis and necrosis. This study is to quantify the morphological change in cell apoptosis induced by H2O2 and investigate a method of diffraction-imaging-flow-cytometry (DIFC) for rapid assay of cells undergoing apoptosis.

Methods: Human leukemia cells (HL-60) were cultured at 37degree celsius in humidified 5percent CO2 atmosphere in RPMI1640 with 10percent heat-inactivated FCS. They were divided into six equal portions (one control, five treated with 1.5mM H2O2) and analyzed at post-treatment time of 0h, 3h, 6h, 12h and 24h, respectively, after synchronization through serum deprivation for 6 hours.flow cytometry (FC) analysis: HL-60 cells were stained with AnnexinV-FITC/PI and analyzed by a BD flow cytometer; quantitative morphology analysis: The cells were imaged with an Olympus inverted-fluorescence-microscopy (IVFM) after stained with Hoechst33342 and AnnexinV-FITC/PI. Morphological parameters were extracted and statistically analyzed with Xcellence-Imaging-Workstation; DIFC analysis: The cells were analyzed without any stains in an in-house-developed DIFC system after resuspended with medium. Images were analyzed by GLCM based software to extract feature parameters as the 3D-fingerprint features.

Results: The FC data clearly differentiated the intact cells at each apoptotic stage for the experimental groups. Fig.1 shows the typical triple-staining fluorescence microscopic images at different apoptotic stage. Some morphological parameters are listed in Table1. Fig.2 shows 4 DIFC images of treated cells. The difference between intact/early apoptotic cells and shrinking cells or fragments can be seen clearly.

Conclusion: The results show that the texture parameters obtained from DIFC measurements correlate well with morphological changes quantified with conventional FC analysis and microscopic measurement in apoptotic cells , which demonstrates that DIFC can provide fast and label-free tool for the study of various cellular processes with underlying morphological changes.corresponding:y_m_feng@yahoo.com

Funding Support, Disclosures, and Conflict of Interest: National Science Foundation of China(NSFC-81171342,81041107)